Method for stabilizing rubella HA antigen

ABSTRACT

A method for stabilizing rubella HA antigen which comprises adjusting a rubella HA antigen suspension at pH 9.6 or higher, and more preferably, concurrently adding sodium azide thereto, or adding sodium azide alone at a concentration of 1 to 10% (w/v) thereto without the above adjustment of pH.

BACKGROUND OF THE INVENTION

1. Field of the Invention

A certain kind of virus carries a component called HA antigen(Hemagglutination Antigen), which has a property agglutinating theanimal erythrocytes, on their particle surface. Anti-virus antibodiesinhibit the agglutination between the HA antigens and the animalerythrocytes. An anti-virus antibody titer can be determined by theHemagglutination Inhibition (HI) test utilizing such a property of HAantigen.

Of the clinical tests for various viruses, the most general one isdetermination of antibody titer against rubella virus; and the HI testis generally used as serological method for diagnosing an infection oranamnetic infection with rubella virus. Particularly, rubella infectionin the first pregnant stage is a matter of primary concern for pregnantwomen in a delivery stage as it causes a birth of congenital rubellachild, so that the rubella HI antibody titer test has widely beenapplied as one of screening tests for pregnant women.

This invention relates to a method for stabilizing rubella HA antigenwhich is used in the rubella HI test based on the hemagglutinationinhibition.

2. Prior Art

Rubella HA antigen is used in the rubella HI antibody titer testpracticed generally. Rubella HA antigen suspension is desired to bestable without deterioration of antigen titer over a long term forsimplifying a titer-adjusting procedure at the time of use and also forsecuring reproducibility of the data and accuracy of the test. And thestabilization prevents the waste of the reagent especially in dealingwith a small number of samples. Therefore, various methods for keepingrubella HA antigen stable have been studied. For example, P. E. Halonenreported that rubella HA antigen which was extracted from the cellsinfected by rubella virus with a glycine buffer (pH 9.0) was stable at4° C. and -70° C. for a few weeks. [Proc. Soc. Exp. Biol. Med. 125,162-167 (1967)]. It is advertised that commercially available rubella HAantigen (by Flow Co.) is stable at pH 9.0-9.5 at 4° C. for 24 hours, butunstable at the lower pH range. Addition of sodium azide as a sterilizerat a concentration of about 0.1% is also effective.

SUMMARY

A rubella HA antigen suspension can be stabilized by adjustment at pH9.6 or higher, preferably pH 9.7 to 11.0 and more preferably about pH10. In addition to the above pH adjustment, the rubella HA antigensuspension is further stabilized by addition of sodium azide at aconcentration of 0.05-10% (w/v). Sodium azide acts bacteriostatically ata concentration of above 0.05% and as a stabilizer at a concentrationabove 1%. Sodium azide, alone, also stabilizes the suspension at aconcentration of 1-10% (w/v) without the adjustment of pH.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the influence of pH range on stability of rubella antigen.

FIG. 2 shows the influence of concentration of sodium azide on stabilityof rubella antigen, and

FIG. 3 shows the influence of pH range and concentration of sodium azideon stability of rubella antigen. In each figure the horizontal axismeans the stored days and the vertical axis means rubella HA antigentiter.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Various methods for stabilizing rubella HA antigen have been studied butsuch a method to fulfill the above requirements for stabilization hasnot yet been developed. The inventors of the present application firstinvestigated the influence of pH of an antigen suspension on itsstability in developing a longterm-stable rubella HA antigen suspensionand elucidated that the rubella HA antigen suspension is not stable at aconventional pH range but more stable at higher pH. Moreover, inaddition to about 0.1% of sodium azide added to the suspension in orderto prevent the decrease of HA antigen titer by contamination ofmicroorganisms during preservation, adjustment of pH and concurrentaddition of an appropriate additional amount of sodium azide in such away as noted above make the HA antigen stable synergistically.

A rubella HA antigen suspension can be stabilized by adjustment at pH9.6 or higher, preferably pH 9.7 to 11.0 and more preferably about pH10. This adjustment of pH can be achieved by using usual bases such assodium hydroxide, potassium hydroxide, aluminium hydroxide and so on.The rubella HA antigen suspension may be lyophilized as necessary andthe adjustment of pH may be carried out before or after lyophilization.The pH fixed before lyophilization does not alter after the regenerationof the lyophilized suspension and the suspension is kept stable. Inaddition to the pH adjustment, the rubella HA antigen suspension isfurther stabilized by addition of sodium azide at a concentration of0.05-10% (w/v). Sodium azide acts bacteriostatically at a concentrationof above 0.05% and as a stabilizer at a concentration at above 1%.Sodium azide alone also stabilizes the suspension at a concentration of1-10% (w/v) without the adjustment of pH.

The rubella HA antigen suspension of the present invention is so stablethat its antigen titer is not decreased over a long time, compared withconventional suspensions of the prior art. Since the pH and the additionof sodium azide in this invention do not influence any antibody titer inthe rubella HI antibody titer test, the rubella HA antigen suspension ofthis invention can be used in the same manner as conventionalsuspensions.

The following examples are provided to illustrate the embodiment of thisinvention. They are not intended to limit the scope of the invention.

EXAMPLE 1

BHK-21 (Baby hamster kidney cells) are cultured in culture bottles andinfected with rubella virus M-33 strain. After the infected cells arecultured at 37° C. for 3 to 7 days, the resulting culture medium iscollected. The collected rubella HA antigen suspension (500 ml) isinactivated and adjusted to pH 10.0 with 2N-sodium hydroxide. Theresulting precipitate is removed by filtration or centrifugation to givea rubella HA antigen suspension which exhibits pH 10. If required, thesuspension is moved into and lyophilized in each vial at such a volumethat, when the lyophilizate is dissolved with 2 ml/vial of purifiedwater, the resulting suspension shows a titer of about 80 times.

The rubella HA antigen suspension showing pH above 9.6 is prepared andlyophilized in the same procedure as mentioned above.

EXAMPLE 2

To the suspension of rubella HA antigen (pH 10) prepared in Example 1 isadded sodium azide in such a way that the sodium azide concentration is0.1% (w/v) when the suspension is regenerated from the lyophilizate.Thus a pH 10 suspension of rubella HA antigen containing sodium azide isprepared. If necessary, the suspension is lyophilized in the same manneras mentioned in Example 1.

The suspension of rubella HA antigen of which the pH range is 9.6 orhigher and the sodium azide concentration is between 0.05 and 10% (w/v)is prepared in the same procedure as mentioned above. The suspension maybe lyophilized if necessary.

EXAMPLE 3

The rubella HA antigen suspension is collected in the same manner asmentioned in Example 1, and an appropriate amount of sodium azide isadded thereto so as to show 1-10% (w/v) sodium azide concentration.

Method of Collection of Rubella HA Antigen

BHK-21 is cultured in a culture bottle, infected with a rubella virusM-33 strain and incubated at 37° C. On the 3rd to 7th day the culturedmedium is collected, and the infectious virus is inactivated to give arubella HA antigen suspension.

Method of Titration of Rubella HA Antigen (HA Titer)

Titration of rubella HA antigen is carried out according to the methodof National Institute of Health Japan, that is, Veronal buffer [VBS(+)]containing 0.1% bovine serum-albumin (BSA) and 0.005% gelatin is usedfor diluent of serum and antigen. On a microtiter U-plate, the HAantigen is diluted by 2 fold serial dilution. Further 0.25% gooseerythrocytes is added to the diluted antigen, and the mixture is wellshaken, and allowed to stand in a refrigerator. The HA titer of theantigen is defined as the maximum dilution frequency causing completehemagglutination after about an hour.

EXPERIMENT 1

To the collected rubella HA antigen was added 2N-sodium hydroxide toadjust the pH range at from 7.5 to 12. The stability of the HA titer wasmeasured at each pH range of the antigen suspension stored at 4° C. Theresults are shown in Table 1. The antigen suspension adjusted to pH 10.0showed no decrease in the titer for about 2 months, while the antigensuspensions adjusted to pH 9.5 or lower and to pH 12 were unstable.

EXPERIMENT 2

After the collected rubella HA antigen is lyophilized and suspended inpurified water, the suspension is adjusted to pH 10.0 with 2N-sodiumhydroxide and stored at 4° C. In this case the HA titer does notdecrease for at least one month.

EXPERIMENT 3

The rubella HA antigen adjusted to pH 10.0 in Example 1 is lyophilized,suspended in purified water, and the suspension is stored at 4° C. Inthis case the HA titer does not decrease for at least 2 months.

EXPERIMENT 4

To the collected rubella HA antigen is added sodium azide at aconcentration of 0-20% (w/v) and the suspensions are adjusted at a pHrange of from 7.5 to 8.0 and stored at 4° C. The HA titer for eachantigen suspension is measured. The results are shown in FIG. 2. In thiscase the suspensions are stable even at pH 7.5-8.0, at which the HAtiter is easily decreased, for about 2 weeks when 5-10% of sodium azideis added. In addition, the suspensions containing 1-20% of sodium azideare more stable than the suspensions containing no sodium azide.

EXPERIMENT 5

The collected rubella HA antigen is adjusted to pH 10.0 or pH 7.5 with2N-sodium hydroxide. Each antigen suspension is divided into twoportions. Sodium azide is added to one portion at the concentration of5%, but not to another portion. At this time, the pH range of thesuspensions containing sodium azide are altered from 10.0 to 9.7 andfrom 7.5 to 7.7, respectively. These antigen suspensions are stored at4° C. The HA titer of each suspension is measured and the results areshown on FIG. 3. The antigen suspension containing 5% sodium azide at pH10.0 does not show any decrease of HA titer while stored at 4° C. for140 days, and is more stable than the other stabilized antigensuspensions.

EXPERIMENT 6

The rubella HA antibody titer is measured with the stabilized HA antigenas mentioned above. HI antibody titration is carried out according tothe method of National Institute of Health, Japan. The same instrumentsand reagents which are employed in the microtiter method for the HAtitration are used. The maximum serum dilution frequency able to inhibitHA antigen is defined as the antibody titer. The results are shown inthe following Table 1.

                                      TABLE 1                                     __________________________________________________________________________    Titration of HI antibody by the use of stabilized HA antigen                  Found values of rubella HI antibody titer                                     HA antigen                                                                          *Controlled positive serum of                                                                *Controlled positive serum of                                                                *Controlled negative serum of             suspension                                                                          patients (16 times)                                                                          patients (64-128 times)                                                                      patients (<8 times)                       __________________________________________________________________________    pH 9.0                                                                              16             128            --                                        pH 10.0                                                                             16             128            <8                                        pH 11.0                                                                             <8             32             --                                        pH 12.0                                                                             <8             <8             --                                        NaN.sub.3 5%                                                                        16             64             --                                        NaN.sub.3 10%                                                                       16             64             --                                        NaN.sub.3 20%                                                                        8             64             --                                        NaN.sub.3 30%                                                                        8             32             --                                        __________________________________________________________________________     *Controlled positive serum and negative serum of patients mean the serum      of which the HI titer has been previously determined by the HI test using     conventional HA antigen.                                                 

As shown in Table 1, HI antibodies show lower titer when the antigensuspension is adjusted at above pH 11, or when sodium azide is added ata concentration above 20%. But, when the stabilized antigen suspensionis adjusted to its optimum pH of 10.0 and sodium azide is added at aconcentration of 5%, there is no influence on HI antibody titer so thatit can be used in the same manner as usual antigen.

As mentioned above, through the method of stabilization of rubella HAantigen in this invention, the antigens can be utilized effectively andwaste of the can be prevented so that the test can be easily carried outin a laboratory dealing with a very small amount of sample and that thereproducibility and accuracy of the results can be secured.

What is claimed is:
 1. A method for stabilizing inactivated rubella HAantigen which comprises adjusting an inactivated rubella HA antigensuspension at pH 9.6 or higher.
 2. The method as claimed in claim 1,wherein the pH is fixed at 9.7 to 11.0.
 3. The method as claimed inclaim 1, wherein the pH is fixed at about
 10. 4. The method as claimedin claim 1, wherein the rubella HA antigen suspension is adjusted at pH9.6 or higher and sodium azide is added thereto.
 5. The method asclaimed in claim 4, wherein the sodium azide is added at a concentrationof 0.05 to 10% (w/v).
 6. A method for stabilizing inactivated rubella HAantigen which comprises adding sodium azide to an inactivated rubella HAantigen suspension at a concentration of 1 to 10% (w/v).
 7. The methodas claimed in claim 2, wherein the rubella HA antigen suspension isadjusted at pH 9.7 to 11 and sodium azide is added thereto.
 8. Themethod as claimed in claim 3, wherein the pH of the rubella HA antigensuspension is fixed at about 10 and sodium azide is added thereto.